PONE-D-24-32616Targeted metabolomics investigation of metabolic markers of Mycobacterium tuberculosis in the cerebrospinal fluid of paediatric patients with tuberculous meningitisPLOS ONE

Dear Dr. Mason,

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: appreciate the inquisitiveness of the authors in searching for MTB-specific biomarkers in CSF. However, I am curious about the rationale behind selecting a control group for this study. While I understand that a control group can help determine what is specific to MTB, this specificity seems to be evident from the results. Could the authors clarify the necessity and role of the control group in this context?

Reviewer #2: Title: Consider emphasizing the novelty or impact of the study in the title. For instance, adding a phrase like "A Novel Approach to Biomarker Discovery" could enhance its appeal.

Abstract: Additionally, mentioning the potential clinical applications in the abstract would strengthen its relevance.

Study objective: Did you consider framing the objective in a hypothesis-driven manner, which could make it more focused and testable? Given the hurdles in pediatric TB diagnosis and treatment.

Study design: Retrospective - not sure whether you sifted through consent forms where approval was granted for future studies or you consented afresh (it is not clear when reading - make it concise how you accessed CSF samples from 12 year olds and under). Explain how potential confounding factors (e.g., co-infections, treatment history) were controlled or accounted for.

Data analysis: Why was bivariate analysis skipped? Was it considered by the study team even with small sample size?

Results: Consider reorganizing the results to flow more logically from general findings to specific metabolic markers. This will help guide the reader through the data more smoothly. For example you have started with multivariate then univariate analysis yet it should be the other way round. Clearly distinguish between statistically significant findings and trends that may require further investigation.

Discussion: The potential for these markers to be used in clinical settings, such as in diagnostic tests or monitoring disease progression. Providing a practical application of the findings will enhance the study's impact. Discuss the need for validation in larger, more diverse prospective cohorts. Additionally, explore how these findings compare to similar studies in adult populations, if applicable.

Conclusion: Consider providing more specific suggestions for future studies, such as longitudinal studies to assess the temporal dynamics of these metabolic markers or exploring their relevance in other forms of tuberculosis. Reiterate the study’s potential clinical impact more strongly, emphasizing how it could contribute to earlier diagnosis or better treatment strategies for pediatric patients with tuberculous meningitis.

Final thoughts: This manuscript makes a meaningful contribution to the field of pediatric infectious diseases and metabolomics. With minor adjustments, as flagged for the attention of the authors the manuscript can be significantly strengthened. These findings have the potential to pave the way for future research and clinical applications, making it a valuable addition to the literature on tuberculous meningitis.

Reviewer #3: In this manuscript, Victory Samuel and Shayne Mason explore novel TBM diagnosis biomarker using targeted metabolomics analysis of CSF sample from patients with TBM and non-meningitis. They included a 6-targeted metabolites list (a 6x46 dataframe) to perform PCA and PLSDA analysis, T test, and ROC analysis. The authors claimed that nonanoic acid’s existence in the CSF indicated its specifically derived from Mtb, and their short of detection of expected metabolites highlighted the adequate sample preparation. Overall, this manuscript provides a moderate targeted metabolites analysis dataset of the CSF samples. The manuscript could be improved in a few aspects, which are listed below:

1. The analysis largely depends on MetaboAnalyst, which requires a pertinent citation.

2. It is important to mention all the supplementary figures in appropriated sections in the manuscript.

3. The figure legends are too brevity to provide enough information for the readers. Please revise these legends.

4. For the unpaired t test used in Fig.3, the FDR adjusted p value is q value, in fact. Please correct the section 2.4.5, “a p-value <0.05 was” should be “a q-value <0.05”.

5. For the ROC analysis, the authors need comment their analysis results, such as whether the AUC values indicate a rationale for biomarker kit development? And the authors could collectively perform 2,3,4 markers-combined ROC analysis to discriminate the TBM and control group.

6. See page 14 Section 3.2, is it Table 1 or Figure 1, and how many compounds, 9 or 10?

7. The biology of the 4 statically significant metabolites seems beyond the aim of this study, as the aim t is discover novel biomarkers from CSF samples. The reviewer suggested a simple summary is enough.

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Reviewer #1: No

Reviewer #2: Yes: Dr. Steve Wandiga

Reviewer #3: Yes: Rui Yang

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Targeted metabolomics investigation of metabolic markers of Mycobacterium tuberculosis in the cerebrospinal fluid of paediatric patients with tuberculous meningitis

PONE-D-24-32616R1

Dear Dr. Mason,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Guocan Yu

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Yes

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: Yes

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4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: Adjustments are appropriate and for elements that were not possible in this work your team can consider those comments in the next paper

Reviewer #3: This new version provides some updates and fixes some misleading mentioned before. However, I recommend the author that "FDR adjusted p value" is "q value". Thus, " For the univariate measure, a p-value < 0.05

was considered statistically significant" , the "p-value" in fact is "FDR p-value", which also called "q value". I think, " For the univariate measure, a FDR p-value < 0.05 (or q value <0.5) was considered statistically significant" is the right one.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Steve Wandiga

Reviewer #3: Yes: Rui Yang

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